Haptoglobin treatment prevents cell-free hemoglobin exacerbated mortality in experimental rat sepsis,Intensive Care Medicine Experimental

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Haptoglobin treatment prevents cell-free hemoglobin exacerbated mortality in experimental rat sepsis
Intensive Care Medicine Experimental Pub Date : 2021-05-03 , DOI: 10.1186/s40635-021-00387-7
Christian A. Schaer , Victor Jeger , Thomas Gentinetta , Donat R. Spahn , Florence Vallelian , Alain Rudiger , Dominik J. Schaer

Sepsis is a dysregulated host response to infection leading to organ dysfunction, organ failure, and death. Multiple mechanisms promote hemolysis during sepsis, such as complement activation, disseminated intravascular coagulation, hemolytic pathogens, sepsis-induced erythrocyte dysfunction, blood transfusion, and medical procedures with extracorporeal circulation (e.g., renal replacement therapy) [1]. Clinical observations suggested that hemolysis with increased cell-free hemoglobin (CFHb) in plasma correlated with reduced survival in sepsis patients [2,3,4]. CFHb is a toxin, which may worsen sepsis pathophysiology by nitric oxide depletion, oxidative tissue injury, activation of coagulation and innate immune pathways, and as an iron source for pathogens [5]. The acute phase protein haptoglobin is the archetypical Hb scavenger in plasma and irreversibly neutralizes the toxicity of bound Hb [1].

Here, we performed a prospective, randomized, blinded animal study to provide direct experimental evidence that CFHb exacerbates sepsis mortality and test whether haptoglobin administration could revert this potentially detrimental adverse effect of hemolysis. For this, we used a fluid resuscitated fecal peritonitis model in awake rats that we have characterized in detail earlier (Fig. 1a) [6]. We first validated an Hb-administration protocol in 36 septic rats randomized to saline, CFHb, or Hb–haptoglobin infusion. Three hours after a bolus followed by continuous infusion, the mean total Hb concentrations in plasma were 5.4 μM (SD ± 3.2 μM) in the saline group and 30.4 μM (SD ± 17.3 μM) in the Hb group (Fig. 1b). These data confirmed that our infusion protocol resulted in plasma concentrations within the range of CFHb observed in patients with severe sepsis [2, 4]. Co-administration of human plasma-derived haptoglobin prevented Hb's renal clearance, resulting in higher concentrations than in the CFHb group (54.7 μM ± 63.0 μM). We determined the fractions of CFHb, Hb–haptoglobin complexes, and heme-protein adducts by size-exclusion chromatography. CFHb and heme-protein adducts eluting in the albumin region remained suppressed when haptoglobin was administered concomitantly with CFHb. This confirms that Hb remains stabilized in the Hb–haptoglobin complex for prolonged periods in circulation and that the complex efficiently prevents Hb degradation and heme release from CFHb [7, 8].

In the main study, we randomized 54 septic Wistar rats to treatment with saline, CFHb, or Hb–haptoglobin. One animal randomized to the saline group had to be excluded from the study, because the intravenous catheter was dislocated during the experiment. In addition, five non-septic animals were infused with CFHb to exclude acute Hb toxicity in healthy animals. After fecal slurry injection, tachycardia developed in all treatment groups consistent with a systemic inflammatory response (i.e., sepsis) (Fig. 2a). The exact timepoint of animal death was determined based on ECG telemetry recordings. The survival data provided evidence for a significantly higher mortality in the group of septic rats infused with CFHb compared to the septic animals infused with only saline (61% versus 12%; p = 0.0066). Co-administration of haptoglobin with CFHb improved mortality to 17%, which was not significantly different from the saline infusion group (12%) (Fig. 2b).

Previous reports demonstrated that blood transfusion-induced hemolysis caused excess mortality in a canine model of S. aureus pneumonia [5]. In the same model, administration of a haptoglobin concentrate improved shock, lung injury, and survival, suggesting that Hb-scavenging neutralized the adverse effects of CFHb [5]. With our model, we now provide direct evidence that purified Hb administered to reach clinically relevant plasma concentrations acts as a toxin during hemolysis, mimicking the adverse effect of intrinsic hemolysis. Our data collectively suggest that CFHb is a contributor to adverse sepsis outcomes and may provide a rationale for therapeutic haptoglobin supplementation as a strategy to improve clinical sepsis management.

Original data are available upon reasonable request from the corresponding author.

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Rudiger A, Jeger V, Arrigo M, Schaer CA, Hildenbrand FF, Arras M et al (2018) Heart rate elevations during early sepsis predict death in fluid-resuscitated rats with fecal peritonitis. Intensive Care Med Exp 6:28
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Boretti FS, Buehler PW, Dgnillo F, Kluge K, Glaus T, Butt OI et al (2009) Sequestration of extracellular hemoglobin within a haptoglobin complex decreases its hypertensive and oxidative effects in dogs and guinea pigs. J Clin Investig. 119:2271–2280
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Plasma-derived human haptoglobin (mixed phenotype) was provided by CSL-Behring, Bern, Switzerland.

The study was supported by Innosuisse (Grant number 19300.1 PFSL-L) and the Swiss National Science Foundation (Grant number 310030_197823).

Affiliations

Division of Internal Medicine, University and University Hospital Zurich, Raemistrasse 100, 8091, Zurich, Switzerland
Christian A. Schaer, Victor Jeger, Florence Vallelian & Dominik J. Schaer
Institute of Anesthesiology, University and University Hospital Zurich, Raemistrasse 100, 8091, Zurich, Switzerland
Christian A. Schaer, Victor Jeger & Donat R. Spahn
CSL Behring AG, Bern, Switzerland
Thomas Gentinetta
Department of Medicine, Hospital Limmattal, Urdorferstrasse 100, 8952, Schlieren, Switzerland
Alain Rudiger

Authors

Christian A. SchaerView author publications
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Victor JegerView author publications
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Thomas GentinettaView author publications
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Donat R. SpahnView author publications
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Florence VallelianView author publications
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Alain RudigerView author publications
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Dominik J. SchaerView author publications
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Contributions

CAS designed the study, performed experiments, analyzed data, wrote the paper; VJ designed the study, performed experiments, analyzed data; TG analyzed plasma samples; DRS and AR designed the study; FV wrote the paper; DJS designed the study, analyzed data. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Dominik J. Schaer.

Ethical approval and consent to participate

This animal study was approved by the Veterinary Office of the Kanton Zurich, Switzerland.

Consent for publication

Not applicable for this animal study.

Competing interests

The authors declare that they have no competing interests.

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Schaer, C.A., Jeger, V., Gentinetta, T. et al. Haptoglobin treatment prevents cell-free hemoglobin exacerbated mortality in experimental rat sepsis. ICMx 9, 22 (2021). https://doi.org/10.1186/s40635-021-00387-7

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Received: 22 February 2021
Accepted: 13 April 2021
Published: 03 May 2021
DOI: https://doi.org/10.1186/s40635-021-00387-7

中文翻译：

肝红蛋白治疗可防止无细胞血红蛋白加重实验性败血症的死亡率

败血症是宿主对感染的失调反应，导致器官功能障碍，器官衰竭和死亡。多种机制促进败血症期间的溶血，例如补体激活，弥散性血管内凝血，溶血性病原体，败血症诱导的红细胞功能障碍，输血和体外循环的医疗程序（例如肾脏替代疗法）[1]。临床观察表明，血浆中无细胞血红蛋白（CFHb）增加的溶血与脓毒症患者的存活率降低相关[2,3,4]。CFHb是一种毒素，可能通过消耗一氧化氮，氧化组织损伤，激活凝血和先天性免疫途径而使脓毒症的病理生理学恶化，并且是病原体的铁源[5]。

在这里，我们进行了一项前瞻性，随机，盲目的动物研究，以提供直接的实验证据，证明CFHb会加重败血症的死亡率，并测试触珠蛋白的施用是否可以逆转溶血的这种潜在有害不利影响。为此，我们在清醒的大鼠中使用了液体复苏的粪便腹膜炎模型，我们已经对其进行了较早的表征（图1a）[6]。我们首先在随机分配给生理盐水，CFHb或Hb-触珠蛋白输注的36只脓毒症大鼠中验证了Hb的给药方案。推注然后连续输注三个小时后，生理盐水组血浆总Hb平均浓度为5.4μM（SD±3.2μM），Hb组血浆平均Hb浓度为30.4μM（SD±17.3μM）（图1b）。这些数据证实，我们的输注方案导致患有严重败血症的患者的血浆浓度在CFHb范围内[2，4]。人血浆来源的触珠蛋白的共同给药可防止Hb的肾脏清除，从而导致其浓度高于CFHb组（54.7μM±63.0μM）。我们通过尺寸排阻色谱法确定了CFHb，Hb-触珠蛋白复合物和血红蛋白加合物的比例。当触珠蛋白与CFHb并用时，在白蛋白区域洗脱的CFHb和血红蛋白加合物仍被抑制。这证实了血红蛋白在血红蛋白-珠蛋白复合物中可以长期稳定地保持稳定，并且该复合物有效地防止了血红蛋白降解和血红素从CFHb中释放[7，8]。人血浆来源的触珠蛋白的共同给药可防止Hb的肾脏清除，从而导致其浓度高于CFHb组（54.7μM±63.0μM）。我们通过尺寸排阻色谱法确定了CFHb，Hb-触珠蛋白复合物和血红蛋白加合物的比例。当触珠蛋白与CFHb并用时，在白蛋白区域洗脱的CFHb和血红蛋白加合物仍被抑制。这证实了血红蛋白在血红蛋白-珠蛋白复合物中可以长期稳定地保持稳定，并且该复合物有效地防止了血红蛋白降解和血红素从CFHb中释放[7，8]。人血浆来源的触珠蛋白的共同给药可防止Hb的肾脏清除，从而导致其浓度高于CFHb组（54.7μM±63.0μM）。我们通过大小排阻色谱法确定了CFHb，Hb-触珠蛋白复合物和血红蛋白加合物的分数。当触珠蛋白与CFHb并用时，在白蛋白区域洗脱的CFHb和血红蛋白加合物仍被抑制。这证实了血红蛋白在血红蛋白-珠蛋白复合物中可以长期稳定地保持稳定，并且该复合物有效地防止了血红蛋白降解和血红素从CFHb中释放[7，8]。尺寸排阻色谱法检测血红蛋白加合物。当触珠蛋白与CFHb并用时，在白蛋白区域洗脱的CFHb和血红蛋白加合物仍被抑制。这证实了血红蛋白在血红蛋白-珠蛋白复合物中可以长期稳定地保持稳定，并且该复合物有效地防止了血红蛋白降解和血红素从CFHb中释放[7，8]。尺寸排阻色谱法检测血红蛋白加合物。当触珠蛋白与CFHb并用时，在白蛋白区域洗脱的CFHb和血红蛋白加合物仍被抑制。这证实了血红蛋白在血红蛋白-珠蛋白复合物中可以长期稳定地保持稳定，并且该复合物有效地防止了血红蛋白降解和血红素从CFHb中释放[7，8]。

在主要研究中，我们将54只脓毒症Wistar大鼠随机分为生理盐水，CFHb或Hb-触珠蛋白治疗。必须将随机分配到盐水组的一只动物排除在研究之外，因为在实验过程中静脉导管被脱位。此外，向五只非败血性动物注入CFHb，以排除健康动物中的急性Hb毒性。粪便注射后，所有治疗组均出现心动过速，与全身性炎症反应（即败血症）一致（图2a）。根据ECG遥测记录确定动物死亡的确切时间点。存活数据提供了证据，与仅注入盐水的败血症动物相比，注入CFHb的败血症大鼠组的死亡率显着更高（61％比12％；p = 0.0066）。将触珠蛋白与CFHb并用可使死亡率提高至17％，与盐水输注组（12％）无显着差异（图2b）。

先前的报道表明，输血引起的溶血在金黄色葡萄球菌肺炎犬模型中导致过多的死亡率[5]。在同一模型中，施用触珠蛋白浓缩液可改善休克，肺损伤和生存，这表明清除Hb可中和CFHb的不良反应[5]。利用我们的模型，我们现在提供直接的证据，即在溶血过程中，纯化的Hb给药达到临床相关血浆浓度可作为毒素，模仿内在溶血的不利影响。我们的数据共同表明CFHb是导致败血症不良后果的因素，并可能为治疗性结合珠蛋白的补充提供理论依据，以作为改善临床败血症管理的策略。

应相应作者的合理要求，可提供原始数据。

1。
Buehler PW，Humar R，Schaer DJ（2020）血红蛋白疗法和无细胞血红蛋白毒性的区室化。趋势摩尔医学26（7）：683–697
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Janz DR，Bastarache JA，Peterson JF，Sills G，Wickersham N，May AK等人（2013）无细胞血红蛋白，对乙酰氨基酚与败血症患者死亡率之间的关联：一项观察性研究。Crit Care Med 41：784–790
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Janz DR，Bastarache JA，Sills G，Wickersham N，May AK，Bernard GR等人（2013）脓毒症成人触珠蛋白，血红蛋白与死亡率之间的关联。暴击护理。17（6）：R272
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4，
Adamzik M，Hamburger T，Petrat F，Peters J，de Groot H，Hartmann M（2012）严重脓毒症中的游离血红蛋白浓度：测量和结果预测方法。暴击护理16（4）：R125
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5，
Remy KE，Cortés-PuchI，Solomon SB，Sun J，Pockros BM，Feng J等人（2018）肝红蛋白可改善犬肺炎的休克，肺损伤和存活率。JCI透视3（18）：e123013
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6，
Rudiger A，Jeger V，Arrigo M，Schaer CA，Hildenbrand FF，Arras M et al（2018）败血症早期的心率升高预示着液体复苏的粪便性腹膜炎大鼠死亡。重症监护医学Exp 6:28
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7
Boretti FS，Buehler PW，Dgnillo F，Kluge K，Glaus T，Butt OI等人（2009）螯合触珠蛋白复合物中的细胞外血红蛋白降低了其在狗和豚鼠中的高血压和氧化作用。J临床研究。119：2271–2280
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Deuel JW，Vallelian F，Schaer CA，Puglia M，Buehler PW，Schaer DJ（2015）触珠蛋白和血红蛋白的不同靶标特异性定义了针对血管血红蛋白毒性的序贯保护系统。免费Radic Biol Med 89：931–943
CAS文章Google学术搜索

下载参考

血浆来源的人触珠蛋白（混合表型）由瑞士伯尔尼的CSL-Behring提供。

这项研究得到了Innosuisse（批准号为19300.1 PFSL-L）和瑞士国家科学基金会（批准号为310030_197823）的支持。

隶属关系

苏黎世大学附属大学医院内科，瑞士苏黎世Raemistrasse 100，8091
克里斯蒂安·A·舍尔，维克多·杰格，佛罗伦萨·瓦莱利亚和多米尼克·J·舍尔
苏黎世大学附属大学医院麻醉研究所，瑞士苏黎世Raemistrasse 100，8091
克里斯蒂安·A·舍尔（Christian A.Schaer），维克多·杰格（Victor Jeger）和多纳特·R·斯潘（Donat R.Spahn）
CSL Behring AG，伯尔尼，瑞士
托马斯·金蒂内塔
瑞士Schlieren，Urdorferstrasse 100，1995年，利马塔尔医院内科
阿兰·鲁迪格（Alain Rudiger）

作者

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会费

CAS设计研究，进行实验，分析数据，撰写论文；VJ设计了研究，进行了实验，分析了数据；TG分析血浆样品；DRS和AR设计了研究；FV写了这篇论文；DJS设计了研究，分析了数据。所有作者阅读并认可的终稿。

通讯作者

对应于Dominik J. Schaer。

道德上的同意和参与同意

这项动物研究得到瑞士苏黎世坎顿兽医局的批准。

同意发表

不适用于此动物研究。

利益争夺

作者宣称他们没有竞争利益。

发行人的便条

对于已发布地图和机构隶属关系中的管辖权主张，Springer Nature保持中立。

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